Metabolomics, the non-targeted interrogation of small molecules in a biological sample, is an ideal technology for identifying diagnostic biomarkers. Current tissue extraction protocols involve sample destruction, precluding additional uses of the tissue. This is particularly problematic for high value samples with limited availability, such as clinical tumor biopsies that require structural preservation to histologically diagnose and gauge cancer aggressiveness. To overcome this limitation and increase the amount of information obtained from patient biopsies, we developed and characterized a workflow to perform metabolomic analysis and histological evaluation on the same biopsy sample.
Biopsies of ten human tissues (muscle, adrenal gland, colon, lung, pancreas, small intestine, spleen, stomach, prostate, kidney) were placed directly in a methanol solution to recover metabolites, precipitate proteins, and fix tissue. Following incubation, biopsies were removed from the solution and processed for histology. Kidney and prostate cancer tumor and benign biopsies were stained with hemotoxylin and eosin and prostate biopsies were subjected to PIN-4 immunohistochemistry. The methanolic extracts were analyzed for metabolites on GC/MS and LC/MS platforms. Raw mass spectrometry data files were automatically extracted using an informatics system that includes peak identification and metabolite identification software.
Metabolites across all major biochemical classes (amino acids, peptides, carbohydrates, lipids, nucleotides, cofactors, xenobiotics) were measured. The number (ranging from 260 in prostate to 340 in colon) and identity of metabolites were comparable to results obtained with the current method requiring 30 mg ground tissue. Comparing relative levels of metabolites, cancer tumor from benign kidney and prostate biopsies could be distinguished. Successful histopathological analysis of biopsies by chemical staining (hematoxylin, eosin) and antibody binding (PIN-4, in prostate) showed cellular architecture and immunoreactivity were retained.
Concurrent metabolite extraction and histological analysis of intact biopsies is amenable to the clinical workflow. Methanol fixation effectively preserves a wide range of tissues and is compatible with chemical staining and immunohistochemistry. The method offers an opportunity to augment histopathological diagnosis and tumor classification with quantitative measures of biochemicals in the same tissue sample. Since certain biochemicals have been shown to correlate with disease aggressiveness, this method should prove valuable as an adjunct to differentiate cancer aggressiveness.
Aims: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin.
Methods: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin.
Results: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation.
Conclusions: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.
UMFix; methanol; tissue fixation
Prostate cancer is one of the leading causes of cancer related deaths. For diagnosis, predicting the outcome of the disease, and for assessing potential new biomarkers, pathologists and researchers routinely analyze histological samples. Morphological and molecular information may be integrated by aligning microscopic histological images in a multiplex fashion. This process is usually time-consuming and results in intra- and inter-user variability. The aim of this study is to investigate the feasibility of using modern image analysis methods for automated alignment of microscopic images from differently stained adjacent paraffin sections from prostatic tissue specimens.
Tissue samples, obtained from biopsy or radical prostatectomy, were sectioned and stained with either hematoxylin & eosin (H&E), immunohistochemistry for p63 and AMACR or Time Resolved Fluorescence (TRF) for androgen receptor (AR).
Image pairs were aligned allowing for translation, rotation and scaling. The registration was performed automatically by first detecting landmarks in both images, using the scale invariant image transform (SIFT), followed by the well-known RANSAC protocol for finding point correspondences and finally aligned by Procrustes fit. The Registration results were evaluated using both visual and quantitative criteria as defined in the text.
Three experiments were carried out. First, images of consecutive tissue sections stained with H&E and p63/AMACR were successfully aligned in 85 of 88 cases (96.6%). The failures occurred in 3 out of 13 cores with highly aggressive cancer (Gleason score ≥ 8). Second, TRF and H&E image pairs were aligned correctly in 103 out of 106 cases (97%).
The third experiment considered the alignment of image pairs with the same staining (H&E) coming from a stack of 4 sections. The success rate for alignment dropped from 93.8% in adjacent sections to 22% for sections furthest away.
The proposed method is both reliable and fast and therefore well suited for automatic segmentation and analysis of specific areas of interest, combining morphological information with protein expression data from three consecutive tissue sections. Finally, the performance of the algorithm seems to be largely unaffected by the Gleason grade of the prostate tissue samples examined, at least up to Gleason score 7.
Multiplex analysis; Histological sections; Hematoxylin & Eosin; p63/AMACR; Time resolved fluorescence imaging; Image registration; Scale invariant feature transform; Prostate cancer
A novel method for high-throughput proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMA) is described using on-tissue tryptic digestion followed by MALDI imaging MS. A TMA section containing 112 needle core biopsies from lung-tumor patients was analyzed using MS and the data were correlated to a serial hematoxylin and eosin (H&E)-stained section having various histological regions marked, including cancer, non-cancer, and normal ones. By correlating each mass spectrum to a defined histological region, statistical classification models were generated that can sufficiently distinguish biopsies from adenocarcinoma from squamous cell carcinoma biopsies. These classification models were built using a training set of biopsies in the TMA and were then validated on the remaining biopsies. Peptide markers of interest were identified directly from the TMA section using MALDI MS/MS sequence analysis. The ability to detect and characterize tumor marker proteins for a large cohort of FFPE samples in a high-throughput approach will be of significant benefit not only to investigators studying tumor biology, but also to clinicians for diagnostic and prognostic purposes.
Cancer; Formalin-fixed paraffin-embedded; Imaging mass spectrometry; Lung; Tissue
Prostate cancer diagnosis is routinely made by the histopathological examination of formalin fixed needle biopsy specimens. Frequently this is the only cancer tissue available from the patient for the analysis of diagnostic and prognostic biomarkers. There is, therefore, an urgent need for methods that allow the high-throughput analysis of these biopsy samples using immunohistochemical (IHC) markers and fluorescence in situ hybridisation (FISH) analysis based markers.
A method that allows the construction of tissue microarrays (TMAs) from diagnostic prostate needle biopsy cores has previously been reported. However, the technique only allows the production of low-density biopsy TMAs with a maximum of 20 cores per TMA. Here two methods are presented that allow the rapid and uniform production of biopsy TMAs containing between 54 and 72 biopsy cores. IHC and FISH techniques were used to detect biomarker status.
Biopsy TMAs were constructed from prostate needle biopsy specimens taken from 102 patients entered into an active surveillance trial and 201 patients in a radiotherapy trial. The detection rate for cancer in slices of these biopsy TMAs was 66% and 79% respectively. Slices of a biopsy TMA prepared from biopsies from active surveillance patients were used to detect multiple IHC markers and to score TMPRSS2-ERG fusion status in a FISH-based assay.
The construction of biopsy TMAs provides an effective method for the multiplex analysis of IHC and FISH markers and for their assessment as prognostic biomarkers in the context of clinical trials.
Aims: To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin’s fixed prostate biopsies.
Methods: Archival Bouin’s fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and α methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin’s fixed tissues to check its suitability for nuclear morphometry.
Results: MCM-2 staining was completely negative in Bouin’s fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin’s fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin’s fixed tissue. Feulgen staining was also very weak in Bouin’s fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin’s and formalin fixed tissues.
Conclusions: Bouin’s fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin’s fixed biopsies is essential.
Bouin’s fixative; immunohistochemistry; prostate
Multiple biomarker testing is necessary to facilitate individualized treatment of lung cancer patients. More than 80% of lung cancers are diagnosed based on very small tumor samples. Often there is not enough tissue for molecular analysis. We compared three minimal invasive sampling methods with respect to RNA quantity for molecular testing.
106 small biopsies were prospectively collected by three different methods forceps biopsy, endobronchial ultrasound (EBUS) guided transbronchial needle aspiration (TBNA), and CT-guided core biopsy. Samples were split into two halves. One part was formalin fixed and paraffin embedded for standard pathological evaluation. The other part was put in RNAlater for immediate RNA/DNA extraction. If the pathologist confirmed the diagnosis of non-small cell lung cancer(NSCLC), the following molecular markers were tested: EGFR mutation, ERCC1, RRM1 and BRCA1.
Overall, RNA-extraction was possible in 101 out of 106 patients (95.3%). We found 49% adenocarcinomas, 38% squamouscarcinomas, and 14% non-otherwise-specified(NOS). The highest RNA yield came from endobronchial ultrasound guided needle aspiration, which was significantly higher than bronchoscopy (37.74±41.09 vs. 13.74±15.53 ng respectively, P = 0.005) and numerically higher than CT-core biopsy (37.74±41.09 vs. 28.72±44.27 ng respectively, P = 0.244). EGFR mutation testing was feasible in 100% of evaluable patients and its incidence was 40.8%, 7.9% and 14.3% in adenocarcinomas, squamouscarcinomas and NSCLC NOS subgroup respectively. There was no difference in the feasibility of molecular testing between the three sampling methods with feasibility rates for ERCC1, RRM1 and BRCA1 of 91%, 87% and 81% respectively.
All three methods can provide sufficient tumor material for multiple biomarkers testing from routinely obtained small biopsies in lung cancer patients. In our study EBUS guided needle aspiration provided the highest amount of tumor RNA compared to bronchoscopy or CT guided core biopsy. Thus EBUS should be considered as an acceptable option for tissue acquisition for molecular testing.
Accurate neuroendocrine neoplasia (NEN) staging is vital for determining prognosis and therapeutic strategy. The great majority of NENs express chromogranin A (CgA) which can be detected at a protein or transcript level. The current standards for lymph node metastasis detection are histological examination after Hematoxylin and Eosin (H&E) and CgA immunohistochemical (IHC) staining. We hypothesized that detection of CgA mRNA transcripts would be a more sensitive method of detecting these metastases.
We compared these traditional methods with PCR for CgA mRNA extracted from formalin fixed paraffin embedded slides of lymph nodes (n = 196) from small intestinal NENs, other gastrointestinal cancers and benign gastrointestinal disease. CgA PCR detected significantly more NEN lymph nodes (75%) than H&E (53%) or CgA IHC (57%) (p = 0.02). PCR detected CgA mRNA in 50% (14 of the 28) of SI-NEN lymph nodes previously considered negative. The false positive rate for detection of CgA mRNA was 19% in non-neuroendocrine cancers, and appeared to be due to occult neuroendocrine differentiation or contamination by normal epithelium during histological processing.
Molecular pathological analysis demonstrates the limitations of observer-dependent histopathology. CgA PCR analysis detected the presence of CgA transcripts in lymph nodes without histological evidence of tumor metastasis. Molecular node positivity (stage molN1) of SI-NEN lymph nodes could confer greater staging accuracy and facilitate early and accurate therapeutic intervention. This technique warrants investigation using clinically annotated tumor samples with follow-up data.
Chromogranin A; Immunohistochemistry; Histopathology; Lymph node; Metastasis; Micrometastasis; Neuroendocrine tumor; Neuroendocrine neoplasm; RNA; Staging
Cervical cancer has a major impact on developing countries, where screening programs are not well established or effective.
This study aims to investigate Human papillomavirus (HPV) 6, 11 and 18 expression in cervical biopsies by immunohistochemistry, (IHC) followed by typing of high-risk HPV 16 and 18 in high-grades by polymerase chain reaction (PCR).
Settings and Design:
During the study period of six months, 30 biopsy samples were obtained from patients attending various gynecology clinics in and around Trichy District, Tamil Nadu, between January and June 2009.
Materials and Methods:
The ecto- and endoscopic biopsy specimens of the cervix were fixed in 10% buffered formalin; routine paraffin sections were taken for processing and stained with hematoxylin and eosin. The samples were graded as Normal cervicitis, Cervical intraepithelial neoplasia (CIN) I, II, III, and squamous cell carcinoma (SCC), for original diagnosis by pathologists. The extra sections were studied for the expression of HPV 6, 11 and 18 by immunohistochemistry and HPV DNA 16 and18 by PCR.
Out of thirty samples, 15 expressed positive and 15 negative for HPV marker. Twenty-seven cases of cervical gradings have been categorized into high grade CIN II/III, SCC (23) and low grade CIN I (4). The high grades were subjected to PCR for high-risk typing. The results revealed that 15 cases were positive for HPV genotype 16 and eight cases for HPV genotype 18. The prevalence of HPV infection was found to be higher in women aged between 50 and 59.
This study reveals a significant detection of HPV in the South Indian suspected individuals, by the use of advanced techniques such as IHC and PCR.
Cervical intraepithelial neoplasia; human papillomavirus; squamous cell carcinoma; immunohistochemistry; polymerase chain reaction
Capabilities for quantitative mass spectrometry–based proteomic profiling of formalin-fixed archival tissue have been developed. Using Liquid Tissue reagents and protocols, we have effectively profiled and validated known and novel cancer biomarkers across a wide variety of fixed cancer tissue samples. Recently developed applications include quantification of protein expression in fixed archival tissue with an emphasis on oral cavity (head and neck squamous cell carcinoma—HNSCC) and breast cancer. Spectral count bioinformatics provides a non-labeling method for quantitative proteomics of formalin-fixed histologically-defined oral cavity cancer. Results indicate many proteins were differentially expressed in cells obtained by laser microdissection of normal, highly differentiated, moderately differentiated, and poorly differentiated HNSCC fixed tissue. Candidate protein biomarkers found to be differentially expressed in the process of HNSCC progression were confirmed and validated by immunohistochemistry on large panels of HNSCC tissue. In addition, we have developed and evaluated a method for direct detection and absolute quantification by selected reaction monitoring (SRM) of HER2 directly in formalin-fixed paraffin-embedded breast cancer tissue using a stable isotope standard peptide derived from HER2. Soluble protein extracts from a collection of breast cancer tissues known to express a range of HER2 were prepared using Liquid Tissue reagents, and quantitative levels were determined. Results demonstrate the ability to quantitate HER2 expression in Liquid Tissue extracts from fixed tissue sections that correlate with standard IHC and indicate the ability to quantify HER2 in immunohistochemical-negative cells. These cumulative results demonstrate development of technologies for quantitative proteomic analysis of proteins that can be applied to the vast worldwide formalin-fixed tissue archives.
To minimize the necessary number of biopsies for molecular and histological research we evaluated different sampling techniques, fixation methods, and storage procedures for canine liver tissue. For addressing the aim, three biopsy techniques (wedge biopsy, Menghini, True-cut), four storage methods for retrieval of RNA (snap freezing, RNAlater, Boonfix, RLT-buffer), two RNA isolation procedures (Trizol and RNAeasy), and three different fixation protocols for histological studies (10% buffered formalin, RNAlater, Boonfix) were compared. Histological evaluation was based on hematoxylin-eosin (HE) and reticulin (fibrogenesis) staining, and rubeanic acid and rhodanine stains for copper. Immunohistochemical evaluation was performed for cytokeratin-7 (K-7), multidrug resistance binding protein-2 (MRP-2) and Hepar-1.
RNA quality was best guaranteed by the combination of a Menghini biopsy with NaCl, followed by RNAlater preservation and RNAeasy mini kit extraction. These results were confirmed by quantitative RT-PCR testing. Reliable histological assessment for copper proved only possible in formalin fixed liver tissue. Short formalin fixation (1–4 hrs) improved immunohistochemical reactivity and preservation of good morphology in small liver biopsies.
At least two biopsies (RNAlater and formalin) are needed. Since human and canine liver diseases are highly comparable, it is conceivable that the protocols described here can be easily translated into the human biomedical field.
Formalin-fixed prostate biopsies are frequently the only tissue collected at the time of prostate cancer diagnosis. There is therefore a requirement for techniques that allow the use of these prostate biopsy specimens in a high-throughput analysis of immunohistochemical and fluorescence-in-situ-hybridisation-detected biomarkers.
The authors have previously described methods that allow tissue microarray (TMA) construction from prostate biopsies. Here, we describe significant technical innovations that provide an easier and more robust system of biopsy–TMA construction.
Results and discussion
The TMAs produced are of a high density (up to 104 cores each, 8×13) and allow a multiplex analysis of biomarkers in the context of clinical trials.
Prostate cancer; tissue microarrays; biomarkers; needle biopsy; cancer; histopathology; molecular pathology; prostate
Breast cancer is the most common malignancy with the highest incidence rates among women worldwide. Triple-negative breast cancer (TNBC) represents the major phenotype of basal-like molecular subtype of breast cancer, characterized by higher incidence in young women and a very poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs playing significant role in the pathogenesis of many cancers including breast cancer. Therefore, miRNAs are also potential prognostic and/or predictive biomarkers in triple-negative breast cancer patients.
Thirty-nine TNBC patients with available formalin-fixed paraffin-embedded (FFPE) tissues were enrolled in the study. MiR-34a, miR-34b, and miR-34c were analyzed using qRT-PCR and correlated to clinico-pathological features of TNBC patients.
Expression levels of miR-34b significantly correlate with disease free survival (DFS) (p = 0.0020, log-rank test) and overall survival (OS) (p = 0.0008, log-rank test) of TNBC patients. No other significant associations between miR-34a, miR-34b, and miR-34c with available clinical pathological data were observed.
MiR-34b expression negatively correlates with disease free survival and overall survival in TNBC patients. Thus, miR-34b may present a new promising prognostic biomarker in TNBC patients, but independent validations are necessary.
triple-negative breast cancer; miR-34a; miR-34b; miR-34c; prognosis
Diagnosis of prostate cancer (PCa) typically relies on needle biopsies, which are routinely archived in paraffin after formalin fixation and may contain valuable risk or prognostic information. The objective of this study was to determine the feasibility of mRNA and miRNA expression analysis in laser-capture microdissected (LCM) formalin-fixed paraffin-embedded archived prostate biopsies compared to the gold standard of frozen tissue. We analyzed the expression of compartment-specific and PCa-related genes in epithelial and stromal tissues collected from paired sets of archived prostate biopsies and frozen radical prostatectomy specimens from three patients. Our results showed appropriate compartment-specific and PC-related expression with good within patient agreement between the FFPE-biopsies and the frozen tissue. The potential for both mRNA and microRNA expression profiling in the biopsies was also demonstrated using PCR arrays which showed high correlation between the biopsy and frozen tissue, notwithstanding sensitivity limitations for mRNA detection in the FFPE specimen. This is the first study to compare RNA expression from biopsy and frozen tissues from the same patient and to examine miRNA expression in LCM-collected tissue from prostate biopsies. With careful technique and use of appropriate controls, RNA profiling from archived biopsy material is quite feasible showing high correlation to frozen tissue.
prostate cancer; FFPE; RNA expression
About 10-25% of breast cancer patients achieve a pathologically confirmed complete response after neoadjuvant chemotherapy. Tissue samples of pretreatment core biopsies are a valuable resource for translational research aiming towards predictive biomarkers for selecting patients who are likely to benefit from neoadjuvant therapy. The German Breast Group (GBG) and the AGO-B Group (AGO = Working Group Gynecological Oncology) have extensive experience in conducting neoadjuvant clinical trials. Technologies as immunohistochemistry on tissue microarrays and standardized reverse transcription-polymerase chain reaction (RT-PCR) approaches on formalin-fixed paraffin-embedded samples allow high-throughput investigation of protein and mRNA biomarkers. With these approaches, we could demonstrate that molecular tumor subtypes and immunological infiltrates are valuable and independent predictors of therapy response. New biomarkers such as poly(ADPribose) polymerase (PARP) might be useful for the prediction of response to conventional and new targeted therapies. This review summarizes current research projects focusing on biomarker discovery in the neoadjuvant setting.
Neoadjuvant; Chemotherapy; Breast cancer Lymphocytes; PARP
Very few studies have investigated whether the time elapsed between surgical resection and tissue fixation or the difference between core-cut and excision biopsies impact on immunohistochemically measured biomarkers, including phosphorylated proteins in primary breast cancer. The aim of this study was to characterise the differences in immunoreactivity of common biomarkers that may occur (1) as a result of tissue handling at surgery and (2) between core-cuts and resected tumours.
Core-cuts taken from surgical breast cancer specimens immediately after resection (sample A) and after routine X-ray of the excised tumour (sample B) were formalin-fixed and paraffin-embedded and compared with the routinely fixed resection specimen (sample C). The variation in immunohistochemical expression of Ki67, oestrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor 2 (HER2), p-Akt and p-Erk1/2 were investigated.
Twenty-one tissue sets with adequate tumour were available. Median time between collection of core-cuts A and B was 30 minutes (range, 20 to 80 minutes). None of the markers showed significant differences between samples A and B. Similarly, Ki67, ER, PgR and HER2 did not differ significantly between core-cuts and main resection specimen, although there was a trend for lower resection values for ER (P = 0.06). However, p-Akt and p-Erk1/2 were markedly lower in resections than core-cuts (median, 27 versus 101 and 69 versus 193, respectively; both P < 0.0001 [two-sided]). This difference was significantly greater in mastectomy than in lumpectomy specimens for p-Erk1/2 (P = 0.01).
The delay in fixation in core-cuts taken after postoperative X-ray of resection specimens has no significant impact on expression of Ki67, ER, PgR, HER2, p-Akt or p-Erk1/2. However, extreme loss of phospho-staining can occur during routine fixation of resection specimens. These differences are likely attributable to suboptimal fixation and may have major repercussions for clinical research involving these markers.
Immunohistochemical (IHC) staining of formalin-fixed and paraffin-embedded tissues (FFPE) is widely used in diagnostic surgical pathology. All anatomical and surgical pathologists use IHC to confirm cancer cell type and possible origin of metastatic cancer of unknown primary site. What kinds of improvements in IHC are needed to boost and strengthen the use of IHC in future diagnostic pathology practice? The aim of this perspective is to suggest that continuing reliance on immunohistochemistry in cancer diagnosis, search and validation of biomarkers for predictive and prognostic studies and utility in cancer treatment selection means that minimum IHC data sets including “normalization methods” for IHC scoring, use of relative protein expression levels, use of protein functional pathways and modifications and protein cell type specificity may be needed when markers are proposed for use in diagnostic pathology. Furthermore evidence based methods (EBM), minimum criteria for diagnostic accuracy (STARD), will help in selecting antibodies for use in diagnostic pathology. In the near future, quantitative methods of proteomics, quantitative real-time polymerase chain reaction (qRT-PCR) and the use of high-throughput genomics for diagnosis and predictive decisions may become preferred tools in medicine.
Immunoperoxidase; protein lifecycle; surgical pathology; proteomics; evidence based methods; normalization
Background: The isolation of good quality DNA from routinely fixed and processed biopsy samples is crucial for the success of subsequent molecular analysis.
Aims: To compare the amount of β actin DNA extracted from upper gastrointestinal tract biopsies fixed in buffered and unbuffered formalin.
Methods: Amounts of β actin DNA extracted from forceps biopsies of the upper gastrointestinal tract fixed in unbuffered (n = 22) and buffered formalin (n = 16) were estimated by quantitative real-time polymerase chain reaction.
Results: The yield of β actin DNA was significantly higher in biopsies fixed in buffered formalin than in those fixed in unbuffered formalin (median 2.8 × 104 and 5.3 × 102 DNA molecules, respectively; p < 0.005). Furthermore, fixation in buffered formalin led to a more reproducible DNA extraction, as indicated by the coefficient of variation (1.0 and 2.2, respectively).
Conclusions: This study indicates that tissue samples should be fixed in buffered formalin to facilitate the use of molecular pathology analysis in routine biopsy material.
unbuffered formalin fixation; gastric and duodenal biopsy; paraffin wax embedding; real-time (TaqMan) polymerase chain reaction; human β actin
The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer – estrogen receptor and HER2 – at the RNA and protein levels.
Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR.
The immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.
The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.
There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.
Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.
In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.
Despite many attempts to establish pre-treatment prognostic markers to understand the clinical biology of esophageal adenocarcinoma (EAC), validated clinical biomarkers or parameters remain elusive. We generated and analyzed tumor transcriptome to develop a practical biomarker prognostic signature in EAC.
Untreated esophageal endoscopic biopsy specimens were obtained from 64 patients undergoing surgery and chemoradiation. Using DNA microarray technology, genome-wide gene expression profiling was performed on 75 untreated cancer specimens from 64 EAC patients. By applying various statistical and informatical methods to gene expression data, we discovered distinct subgroups of EAC with differences in overall gene expression patterns and identified potential biomarkers significantly associated with prognosis. The candidate marker genes were further explored in formalin-fixed, paraffin-embedded tissues from an independent cohort (52 patients) using quantitative RT-PCR to measure gene expression. We identified two genes whose expression was associated with overall survival in 52 EAC patients and the combined 2-gene expression signature was independently associated with poor outcome (P<0.024) in the multivariate Cox hazard regression analysis.
Our findings suggest that the molecular gene expression signatures are associated with prognosis of EAC patients and can be assessed prior to any therapy. This signature could provide important improvement for the management of EAC patients.
A modified immunofluorescent technique was used for the detection of HBSAg in formalin-fixed liver tissue, thereby allowing retrospective examination of paraffin sections and avoiding the need to split the sample at the time of biopsy. Comparison with two other methods, involving either orcein staining or standard haematoxylin and eosin (H and E) preparation for ground glass hepatocytes, showed slightly fewer positive hepatocytes in individual biopsies with the latter stain, but the specificity of both methods was high. In a series of 146 seropositive and 74 seronegative patients with a variety of liver disorders, hepatocytes positive for HBSAg were found in only one of 55 patients with acute hepatitis type B, whereas large numbers of positive cells were seen in all 22 healthy carriers of the surface antigen. In the 69 patients with chronic persistent or chronic aggressive hepatitis, the frequency of positive biopsies was 86% and 85% respectively. The positive cells in these cases were, in comparison with healthy carriers of HBSAg, much fewer in number and were scattered in random fashion throughout the lobule rather than occurring in discrete clumps or sheets. This variation in the intrahepatic expression of HBSAg may reflect differences in the immune response to hepatitis B viral antigens.
We evaluated the risk of sampling errors in specimens of biopsy size, which may be caused by heterogeneous overexpression of Her2/neu in gastric cancer (GC).
Patients and methods
The study cohort comprised 454 gastrectomy patients with adenocarcinoma of the stomach or esophago-gastric junction. Tissue micro-arrays (TMAs) served as ‘biopsy procedure’ and were generated from formalin-fixed and paraffin-embedded tissue: five tissue cylinders were collected randomly from each tumor, rendering 2230 core cylinders. These were compared with 454 whole tissue sections obtained from the same paraffin blocks. Her2/neu expression and gene amplification were analyzed by immunohistochemistry and in situ hybridization. The Her2/neu status was determined according to GC scoring system by two independent observers.
In whole tissue sections, 37 (8.1%; observer 1) and 38 (8.4%; observer 2) of the GCs, and in the corresponding TMAs, 28 (6.3%; observer 1) and 28 (6.3%; observer 2) of the GCs were classified as Her2/neu-positive (kappa value 98.5% and 96.2%; P < 0001). Comparison of whole tissue sections with corresponding TMAs showed a false-negative rate of 24% and a false-positive rate of 3% for TMAs.
Assessment of the Her2/neu status in tissue biopsies carries a significant risk of sampling errors, thereby rendering patients unsuitable for treatment with trastuzumab.
biopsy; gastric cancer; Her2/neu; sampling error
Fine needle aspiration biopsy (FNAB) is gaining increasing popularity in the diagnosis of musculoskeletal lesions; and in many patients, a definitive diagnosis can be rendered from aspiration smears alone. Its applicability in bone pathology, however, has been controversial due to a high percentage of inadequate smears, difficulty in evaluation of tissue architecture and nonspecific results in the diagnosis of primary bone lesions. In this study, the value of aspiration as the first pathological investigation in the diagnosis of a bone lesion was evaluated.
91 cases of clinically suspected cases of bone lesions were aspirated over a period of two years. Direct or cytospin smears were fixed in 95% alcohol and stained by Hematoxylin and Eosin or air-dried and later fixed in methanol for May Grŭnwald Giemsa staining.
Of the 91 patients who were subjected to FNAB, 81 were considered satisfactory and 10.9 % (10) were inadequate\inconclusive for diagnosis. Cyto-histological concordance was obtained in 78.5 % (51/65) patients. Positive and negative predictive values were 87.5% and 97.2 % respectively. Sensitivity as a preliminary diagnostic technique was 93.3%, whereas specificity was 94.5 %. Overall, diagnostic accuracy was 94.2 %. Metastatic lesions were detected with 100% accuracy. Two cases were reported as false positive and one case as false negative.
Cytology provides valuable information to the clinician to make an informed decision regarding appropriate therapy. We conclude that time-consuming and costly investigations may be reduced by choosing FNAB as the initial pathological diagnostic method for skeletal lesions of unknown origin. The choice of radiological examinations, laboratory tests and surgical biopsies can be determined after the FNAB diagnosis.
The extraction of DNA from formalin fixed, paraffin wax embedded tissue can be problematical, with long protocols producing low yields. This report describes a very simple and useful method for amplifying DNA from formalin fixed, paraffin wax embedded tissue without the need for prior DNA extraction. This method allows direct polymerase chain reaction (PCR) based molecular analysis of fixed tissue. It is an invaluable method if clinical biopsy specimens are to be investigated, because extraction of uncontaminated DNA from such small samples can be very difficult or even impossible. It will also facilitate the study of intratumour heterogeneity, with the analysis of multiple small areas from within a single tumour section. In addition, this method can be used for other samples where only a few tests are to be carried out and a stock of DNA is not required, thus shortening the analysis time.
archival tissue; multiplex amplification; DNA extraction